Substantial evidence supports the role for IGF-I signaling in mediating the skeletal response to load. Osteocytes and mature osteoblasts are the likely candidates by which mechanical load is initially transmitted into signals for new bone formation. IGF-I production is increased in these cells following mechanical load, and this IGF-I may be the signal for the production of new osteoblasts. Furthermore, mechanical load enhances the skeletal response to IGF-I just as skeletal unloading leads to resistance to IGF-I with respect to bone formation and osteoprogenitor proliferation. This failure to respond to IGF-I in vivo or in vitro with unloading is due to a failure of IGF-I to activate the IGF-I receptor (IGF-IR), although IGF-IR levels and binding of IGF-I to its receptor are normal. The resistance to IGF-I caused by skeletal unloading can be reversed by reloading the bones in vivo or the osteoblasts in vitro. Integrins play an important role in this process. Skeletal unloading reduces integrin expression, and this inhibition of expression can be restored by reloading the bones commensurate with restoration of IGF-I responsiveness. Co-immunoprecipitation and confocal studies demonstrate direct binding of 1 and 3 integrin subunits to the IGF-IR, binding which is enhanced by IGF-I. Cells grown on specific integrin substrates demonstrate enhanced activation of the IGF-IR by both IGF-I and loading, and enhanced IGF-I activation of the 3 integrin subunit. Knockdown of either the 1 or 3 integrin subunits in osteoblasts blocks the ability of IGF-I to activate its receptor and prevents its activation by loading concomitant with a disruption of downstream IGF-I signaling. Thus, IGF-I signaling is important for the skeletal response to bone, and this signaling is regulated by integrins. In this project we will test the hypothesis that the anabolic response of bone to mechanical load requires the synergistic interaction between integrin and IGF-I signaling. Three aims are proposed: 1. Determine the mechanism for regulation of IGF-I signaling by integrins during mechanical loading, and the extent to which the maturation of the osteoblast modifies this regulation. These studies will be done with osteoblast and osteocyte cell lines mechanically loaded by pulsatile fluid flow (PFF), analyzing the components and roles of the integrin/IGF-IR complex. 2. Determine the requirement for IGF-I and IGF-IR in the skeletal response to mechanical load. These studies will be done with mice in which IGF-I and IGF-IR are deleted from osteoprogenitors and osteoblasts/osteocytes then determining the response to load in vivo and in vitro. 3. Determine the requirement for the 1 and 3 integrin subunits in the skeletal response to mechanical load. These studies will be done by deleting 1 and 3 integrin from osteoprogenitors and osteoblasts/osteocytes then determining their response to load and to IGF-I. Thus using both in vivo and in vitro approaches we will establish the role and importance of integrin/IGF-I signaling in the skeletal response to load, results critical to our understanding of bone loss during skeletal immobilization from a variety of causes including paralysis and prolonged immobilization following injury or sickness. PUBLIC HEALTH RELEVANCE: Skeletal unloading results in the loss of bone which is due in part to failure of bone to respond to the anabolic actions of IGF-1. This failure is associated with loss of integrin expression. This project will evaluate the interaction between integrin and IGF-1 signaling in the skeletal response to mechanical load.